AQ-13, an investigational antimalarial, versus artemether plus lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria: a randomised, phase 2, non-inferiority clinical trial.

BACKGROUND: Chloroquine was used for malaria treatment until resistant Plasmodium falciparum was identified. Because 4-aminoquinolines with modified side chains, such as AQ-13, are active against resistant parasites, we compared AQ-13 against artemether plus lumefantrine for treatment of uncomplicated P falciparum malaria.
METHODS: We did a randomised, non-inferiority trial. We screened men (≥18 years) with uncomplicated malaria in Missira (northeast Mali) and Bamako (capital of Mali) for eligibility (≥2000 asexual P falciparum parasites per μL of blood). Eligible participants were randomly assigned to either the artemether plus lumefantrine group or AQ-13 group by permuting blocks of four with a random number generator. Physicians and others caring for the participants were masked, except for participants who received treatment and the research pharmacist who implemented the randomisation and provided treatment. Participants received either 80 mg of oral artemether and 480 mg of oral lumefantrine twice daily for 3 days or 638·50 mg of AQ-13 base (two oral capsules) on days 1 and 2, and 319·25 mg base (one oral capsule) on day 3. Participants were monitored for parasite clearance (50 μL blood samples twice daily at 12 h intervals until two consecutive negative samples were obtained) and interviewed for adverse events (once every day) as inpatients during week 1. During the 5-week outpatient follow-up, participants were examined for adverse events and recurrent infection twice per week. All participants were included in the intention-to-treat analysis and per-protocol analysis, except for those who dropped out in the per-protocol analysis. The composite primary outcome was clearance of asexual parasites and fever by day 7, and absence of recrudescent infection by parasites with the same molecular markers from days 8 to 42 (defined as cure). Non-inferiority was considered established if the proportion of patients who were cured was higher for artemether plus lumefantrine than for AQ-13 and the upper limit of the 95% CI was less than the non-inferiority margin of 15%. This trial is registered at ClinicalTrials.gov, number NCT01614964.
FINDINGS: Between Aug 6 and Nov 18, 2013, and between Sept 18 and Nov 20, 2015, 66 Malian men with uncomplicated malaria were enrolled. 33 participants were randomly assigned to each group. There were no serious adverse events (grade 2-4) and asexual parasites were cleared by day 7 in both groups. 453 less-severe adverse events (≤grade 1) were reported: 214 in the combination group and 239 in the AQ-13 group. Two participants withdrew from the AQ-13 group after parasite clearance and three were lost to follow-up. In the artemether plus lumefantrine group, two participants had late treatment failures (same markers as original isolates). On the basis of the per-protocol analysis, the AQ-13 and artemether plus lumefantrine groups had similar proportions cured (28 [100%] of 28 vs 31 [93·9%] of 33; p=0·50) and AQ-13 was not inferior to artemether plus lumefantrine (difference -6·1%, 95% CI -14·7 to 2·4). Proportions cured were also similar between the groups in the intention-to-treat analysis (28 of 33, 84·8% for AQ-13 vs 31 of 33, 93·9% for artemether and lumefantrine; p=0·43) but the upper bound of the 95% CI exceeded the 15% non-inferiority margin (difference 9·1%, 95% CI -5·6 to 23·8).
INTERPRETATION: The per-protocol analysis suggested non-inferiority of AQ-13 to artemether plus lumefantrine. By contrast, the intention-to-treat analysis, which included two participants who withdrew and three who were lost to follow-up from the AQ-13 group, did not meet the criterion for non-inferiority of AQ-13, although there were no AQ-13 treatment failures. Studies with more participants (and non-immune participants) are needed to decide whether widespread use of modified 4-aminoquinolones should be recommended. FUNDING: US Food and Drug Administration Orphan Product Development, National Institutes of Health, US Centers for Disease Control and Prevention, Burroughs-Wellcome Fund, US State Department, and WHO.

Evidence before this study

Chloroquine was the treatment of choice for Plasmodium falciparum malaria until chloroquine-resistant parasites were identified more than 50 years ago. We have considered evidence from several studies done in our laboratories and those of colleagues worldwide, beginning in 1981. During this period, we have done many literature searches using different search terms without restriction to language each year. For these reasons, we have not listed the specific search criteria or results. Although chloroquine-resistant P falciparum have continued to be problem, our recent studies have shown that 4-aminoquinolines (4-AQs) with modified side chains, such as AQ-13, are active against chloroquine-resistant parasites and safe in human beings, and have pharmacokinetics similar to chloroquine.

Added value of this study

In this randomised, non-inferiority clinical trial of Malian men with uncomplicated P falciparum malaria, the per-protocol analysis suggested non-inferiority of AQ-13 to artemether plus lumefantrine. By contrast, the intention-to-treat analysis, which included two participants who withdrew and three participants lost to follow-up from the AQ-13 group, did not meet the criterion for non-inferiority of AQ-13, although there were no AQ-13 treatment failures. Adverse events were much the same in each group.

Implications of all the available evidence

Our results indicate that AQ-13 is not inferior to artemether plus lumefantrine for treatment of uncomplicated P falciparum malaria due to chloroquine-resistant and chloroquine-susceptible parasites. Modified 4-AQs, such as AQ-13 might expand the currently limited number of antimalarial drugs active against drug-resistant parasites. Additional studies with non-immune patients and more participants are needed to decide whether to recommend widespread use of modified 4-AQs for uncomplicated malaria.

Introduction

Plasmodium falciparum parasites that are resistant to chloroquine were first identified more than 50 years ago in southeast Asia and South America as it became apparent that treatment of malaria due to chloroquine-resistant P falciparum (defined as 50% inhibitory concentrations [IC50s] >200 nM) often failed in non-immune individuals. Subsequently, chloroquine-resistant P falciparum has spread to east and west Africa, across South America, and to Oceania.

Studies of 4-aminoquinoline (4-AQ) activity against P falciparum have shown that aminoquinolines with side chains shorter (two to three carbons) or longer (ten to 12 carbons) than the five carbon isopentyl side chain of chloroquine are active against chloroquine-resistant P falciparum. For this reason, an Investigational New Drug (IND) application (IND 055,670) was submitted to the US Food and Drug Administration (FDA) to examine the lead compound from those studies (AQ-13 [N′-(7-chloro-4-quinolinyl)-N,N-diethyl-1,3-propanediamine dihydrochloride trihydrate], which has a linear three carbon n-propyl side chain) in healthy human beings to determine if its pharmacokinetics and safety profiles were similar to those of chloroquine.

Because the lead compound, AQ-13, from studies of structure and activity was active in vitro against chloroquine-resistant and chloroquine-susceptible P falciparum, and safe in human participants, we postulated that the efficacy and safety of AQ-13 for uncomplicated P falciparum malaria would not be inferior to that of artemether plus lumefantrine; in this trial we aimed to test the hypothesis.

Methods

Study design and participants

Because the efficacy of artemether with lumefantrine for uncomplicated P falciparum malaria is 95% or more, and because AQ-13 had not been used to treat malaria in people, we used a non-inferiority study design. This study comprised a 1-week inpatient stay and two outpatient follow-up visits every week for the next 5 weeks (total of 6 weeks), and was done at the Clinical Research Center in Bamako, Mali. Participants recruited were Malian men with uncomplicated P falciparum malaria (fever, chills, or other symptoms; and a positive blood smear, with 2000 or more asexual P falciparum parasites per μL of blood). Men aged 18 years or older were enrolled because they are semi-immune and therefore protected from severe disease (appendix). Women were excluded at the request of the FDA to prevent exposure to an investigational drug during pregnancy. Additional exclusion criteria were severe or cerebral malaria, plasmodia infection other than P falciparum, a blood haemoglobin concentration of 7 g/dL or less, medications for chronic diseases or conditions other than malaria, and other health problems requiring diagnosis or treatment.

All participants provided consent. Consent forms were prepared in English and translated to French and viewed and approved by the Tulane and Mali Institutional Review Boards before use. Information in the consent form was read to potential participants by investigators who understood the protocol and could discuss it in local languages.

Ethical reviews and approvals were provided by institutional review boards at the Tulane University (New Orleans, LA, USA; FWA 00002055) and the University of Bamako (Mali; FWA 00001679). The protocol is available online.

Randomisation and masking

Enrolled participants were randomly assigned to either artemether plus lumefantrine or AQ-13. The statistician (FJM) prepared the randomisation by permuting blocks of four with a random number generator. Although the original plan was to provide this information in sealed envelopes, an electronic list was provided instead at the request of the investigators in Mali. The randomisation was implemented in Mali by the research pharmacist (AM) who met twice daily with each participant on days 1–3 of their inpatient stay and observed their treatment. Physicians and others caring for the participants were masked to allocation, except the research pharmacist who provided treatment. Neither the study statistician nor the research pharmacist had other involvement with the performance or supervision of this clinical trial. Patients were not masked to the treatments they received.

Because previous studies have shown more rapid clearance of asexual parasites from the blood with artemisinins than with 4-AQs, such as chloroquine or amodiaquine, we expected parasite clearance times to be shorter with artemether plus lumefantrine than with AQ-13. Therefore, to avoid inadvertent unmasking of the study, parasite clearance times were not compared for the two treatment groups until after the study data were unblinded on Jan 2, 2016.

Procedures

Potential participants were screened for abnormal laboratory results unrelated to malaria using the hepatic and renal disease panels for the Piccolo Xpress chemistry analyzer (Princeton, NJ, USA) and complete blood counts from the Beckman coulter counter (Indianapolis, IN, USA). Enrolled participants randomly assigned to artemether with lumefantrine received 80 mg of oral artemether and 480 mg of oral lumefantrine twice a day for 3 days as recommended in the package insert (Coartem; Novartis Beijing, Beijing, China; Pharma AG, Basel, Switzerland). Those assigned to AQ-13 received 638·50 mg of AQ-13 base (two capsules) on days 1 and 2, and 319·25 mg base (one capsule) on day 3 based on the phase 1 study (appendix). AQ-13 was synthesised under current good manufacturing practice guidelines by Girindus America (Cincinnati, OH, USA) and transported to the study site at 15–30°C.7, 8

During the 5-week follow-up, two outpatient visits per week occurred and interval histories, physical examinations, and blood smears were done. Additionally, a follow-up eye examination on day 7 occurred and 1 h Holter recordings were obtained on days 7 and 14. We used fingerstick blood samples (50 μL obtained twice daily during week 1) until there were two consecutive negative smears to monitor parasite clearance. During the twice weekly outpatient follow-up visits from week 2 to week 6, we examined participants for fever, parasitaemia, and other evidence of recurrence. Clearance and recrudescence were based on Giemsa-stained thick smears plus PCR-based molecular markers for recrudescence.

Blood smears were prepared on glass slides, stained with Giemsa, and stored in slide boxes after reading. Filter paper blots were labelled, placed in individual plastic bags with desiccant, and stored at ambient temperature. Blood samples for haematological testing were drawn in tubes containing heparin and for chemistry testing in tubes containing EDTA (ethylenediaminetetraacetic acid). These samples were transported to the clinical laboratory within 2–3 h. Blood samples for AQ-13 measurements were stored in EDTA at 4°C until the time of analysis.

Most participants had minimal literacy, so they could not use diaries to record adverse events. Therefore, potential events were discussed with each patient by the investigators and clinical centre staff daily during the 1-week inpatient stay and twice per week during the 5-week outpatient follow-up period. Participants were asked to respond to a list of questions on the case report forms. Potential adverse events discussed included fever, weakness, myalgias and arthralgias, headache, anorexia, nausea, vomiting, abdominal pain, diarrhoea, cough, pruritus, tinnitus, influenza-like symptoms, pallor, and jaundice. Participants were encouraged to report all adverse events, not just those listed. Potential participants were examined during screening to identify people with reduced visual acuity or abnormalities of the eyelids, cornea, iris, or retina. After oral treatment on inpatient days 1–3, the eye examination was repeated on day 7 to identify if there had been any change or if there was evidence of ocular toxicity.

Cardiac adverse events, such as arrhythmias and heart block, were detected with Holter monitoring and was done for 1 h during screening, for 24 h each day on days 1–4 of the inpatient stay, and for 1 h during the outpatient follow-up visits on days 7 and 14. These recordings were reviewed on the basis of the QT interval summary report provided by HolterCare (version 10.6.0) for arrhythmias and heart block in Mali and for evidence of QTc (corrected QT) prolongation after treatment with either drug.

In our studies of pharmacokinetics, blood samples for measurements of concentrations of AQ-13 and its primary and secondary metabolites (AQ-72 and AQ-73) were obtained from the participants enrolled in this study and randomly assigned to AQ-13 during 2015 after additional training on drawing timed venous blood samples had been provided to the investigators and staff. Beginning after informed consent was obtained, serial 5·0 mL blood samples were drawn from each participant before treatment began on day 1 (0 time baseline sample), after treatment on days 1–3 (n=21 samples), daily during the remainder of the inpatient stay (n=3), and twice weekly during the subsequent 5-week outpatient follow-up period (n=10, ∑=35; appendix).

To assess parasite genotype for measuring recrudescence, we did sequencing. After nested PCR amplifications using primers for 546 bp and 166 bp amplicons within pfcrt (appendix),16, 17 dideoxynucleotide sequencing was done to identify the pfcrt haplotypes (aminoacids 72–76—ie, CVIET and CVMNK) of the isolates from all participants. Additionally, allotype-specific primers were used to amplify block 2 sequences from merozoite surface protein 1 after initial amplification with universal primers in blocks 1 and 5.

Outcomes

The primary endpoint was clinical cure, a composite of clearance of asexual P falciparum parasites from the blood and fever by day 7, and the absence of recrudescent infection with the same parasite genotype between the time of initial parasite clearance and the final outpatient visit on day 42. The proportions of patients cured were calculated for artemether with lumefantrine and AQ-13, and differences between proportions cured were calculated with 95% CIs. Non-inferiority of AQ-13 was considered established if the proportion cured was higher for artemether plus lumefantrine than for AQ-13 and the upper limit of the 95% CI for the differences between the proportions cured was less than the non-inferiority margin of 15%.

Secondary outcomes included adverse events within 28 days of treatment judged as either possibly, likely, or definitely related to treatment by masked physician-investigators, times from treatment to parasite and fever clearance, mean decrease in the haemoglobin concentration from days 1 to 4, new atrial or ventricular arrhythmias, new first, second, or third degree heart block, prolongation of the QT interval, and decreases in visual acuity. Parasite clearance time was defined as the time of the first persistently negative thick blood smears for asexual P falciparum parasites during the 1 week inpatient stay, and fever clearance time was defined as the first persistently normal temperature (<37·5°C) during week 1 of inpatient stay.

Tertiary outcomes included the pharmacokinetics of AQ-13 N-dealkylated metabolites (AQ-72, AQ-73) based on blood concentrations which were measured using a high-performance liquid chromatography assay developed for this purpose9, 15 and the frequency of pruritus after treatment. Because neither the primary nor the secondary metabolites of AQ-13 are active in vitro against chloroquine-resistant P falciparum, pharmacokinetic parameters potentially related to efficacy have been provided only for AQ-13.

Statistical analysis

Sample size estimates were based on a 15% non-inferiority margin, 95% cure for those given artemether with lumefantrine, and 20% attrition after treatment with the power (1 – β=80%) to detect a 15% or greater decrease in efficacy, which yielded sample sizes of 33 per group and 66 participants in total. Testing of the primary and secondary outcomes was done with the Fisher's exact test, t test, or Mann-Whitney U test. These statistical tests were done in GraphPad Prism (version 6.07). Times to parasite and fever clearance were compared with Kaplan-Meier survival curves and tested for significance using the log-rank test. Pharmacokinetic parameters for AQ-13 from phase 2 efficacy studies were based on non-compartmental analysis with the use of the Win Nonlin software in Pharsight (version 6.5) and compared with t tests for which p values less than 0·05 were considered significant. Two-sided testing was used for all statistical comparisons.

The data and safety monitoring board did an interim analysis after 33 patients were enrolled in the study. This trial was registered at ClinicalTrials.gov, number NCT01614964.

Role of the funding source

The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of this report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit this manuscript for publication.

Results

Participants were enrolled from Aug 6 to Nov 18, 2013, and from Sept 18 to Nov 20, 2015. No participants were enrolled in 2014 because of concern the criterion for screening (fever in the absence of localising signs) could lead to the enrolment of people with Ebola virus disease.

202 individuals in Missira, northeast Mali, and 139 in Bamako were screened. 275 individuals did not meet the inclusion criteria and 66 participants were enrolled. Of those enrolled, half were randomly assigned to artemether plus lumefantrine and the other half were randomly assigned to AQ-13. In the artemether plus lumefantrine group, 33 participants were treated successfully and 31 completed the 42-day follow-up, whereas two had late treatment failures on days 17 and 21. In the AQ-13 group, 33 participants were treated successfully and 28 completed the 42-day follow-up, whereas two withdrew on day 4 for personal reasons and three were lost to follow-up on days 10, 28, and 35. All those given artemether plus lumefantrine were included in the intention-to-treat and per-protocol analyses, whereas only 28 given AQ-13 were included in the per-protocol analysis because two withdrew and three were lost to follow-up (figure 1).

Figure 1

Trial profile

*Individuals with fever, chills, or other symptoms consistent with malaria were screened with blood smears, physical examinations (including temperatures), and previous medical records. †These participants were excluded because they had negative blood smears for asexual Plasmodium falciparum parasites or less than 2000 asexual parasites per μL.

Baseline characteristics were similar in each group (table 1, appendix). Treatment based on randomisation to either intervention group was confirmed by fluorescence high-performance liquid chromatography assays for AQ-13,9, 15 which were positive for participants randomly assigned to AQ-13 and negative for those randomly assigned to artemether plus lumefantrine.

Table 1

Baseline characteristics

	Artemether plus lumefantrine (n=33)	AQ-13 (n=33)
Age (years)	31·9 (14·8)	30·3 (12·2)
Height (cm)	175·5 (7·1)	174·3 (7·2)
Weight (kg)	67·5 (11·7)	64·2 (7·8)
Median number of asexual parasites per μL (IQR)	12 000 (21 575)	11 850 (28 900)
Haemoglobin concentration (g/dL)	12·5 (2·1)	12·1 (1·8)
CVIET* genotype parasites	13 (39·4%)	16 (48·5%)
CVMNK* genotype parasites	14 (42·4%)	12 (36·4%)
CVIET and CVMNK genotype parasites	6 (18·2%)	5 (15·2%)

Data are mean (SD) or n (%), unless otherwise stated. All participants were men, of Malian ethnicity, had haematology panel, chemistry panel, and eye examination screenings, and had Holter recording for sinus rhythm. No participants had first, second, or third degree heart block. Normal ranges for the haematology and chemistry panels are provided in the appendix.

CVIET and CVMNK are the pfcrt haplotypes at aminoacids 72–76.

The components of the primary endpoint (clearance of asexual parasites and fever by day 7) were achieved for all participants in both groups. The third component (absence of recrudescence with the original parasites from days 8 to 42) was achieved for 31 (93·9%) of 33 participants in the artemether plus lumefantrine group and 28 (100%) of 28 in the AQ-13 group (table 2). The per-protocol analyses showed that the proportions cured were greater for AQ-13 than for artemether plus lumefantrine (28 [100%] of 28 vs 31 [93·9%] of 33) with a difference of −6·1% (95% CI −14·7 to 2·4); therefore AQ-13 was non-inferior according to this anaylsis. By contrast, the intention-to-treat analyses showed that the proportions cured were lower for AQ-13 than for artemether plus lumefantrine (28 [84·8%] of 33 vs 31 [93·9%] of 33) with a difference of 9·1% (−5·6 to 23·8), for which the upper boundary of the 95% CI exceeded the non-inferiority margin of 15%.

Table 2

Primary and secondary outcomes

	Artemether plus lumefantrine (n=33)	AQ-13 (n=33)	Relative risk	p value
Primary outcome
Cure (intention-to-treat analysis)	31/33 (93·9%)	28/33 (84·8%)	0·90	0·43
Cure (per-protocol analysis [numbers of participants cured])	31/33 (93·9%)	28/28 (100%)*	1·06	0·50*
Parasite and fever clearance by day 7	33/33 (100%)	33/33 (100%)	1·00	1·00
Recrudescence of infection (days 8–42)	2/33 (6%)	0/28*	0·24	0·50*
Secondary outcomes
Grade 2–4 adverse events	0	0	1·00	1·00
Less serious adverse events (≤grade 1)	33 (100%)	32 (97%)	0·97	1·00
Mean parasite clearance time (h)	32·5 (28·0–37·0)	47·3 (43·5–51·1)	14·8†	0·002
Mean fever clearance time (days)	1·23 (1·08–1·38)	1·00 (1·00–1·00)	−0·23†	0·01
Mean decrease in haemoglobin concentration (days 1–4)	1·5 (0·7–2·3)	0·4 (0·3–0·5)	−1·1†	0·02
New atrial or ventricular arrhythmias	0	0	1·07	1·00
New first, second, or third degree heart block	0	0‡	1·00	1·00
Mean increase in QTc 4 h after dose (ms)	0·4 (−0·4 to 1·1)	−1·2 (−2·9 to 0·6)	−1·6†	0·33
Decreased visual acuity on day 7	0	0‡	1·00	1·00

Data are n (%) or mean (95% CI). On the basis of doing multiple tests, the adjusted (corrected) value of α (αc) was 0·004. QTc=corrected QT.

Two participants who withdrew on day 4 and three who were lost to follow-up were not included in this analysis.

Data provided are the mean for participants randomly assigned to the AQ-13 group minus the mean for participants randomly assigned to the artemether plus lumefantrine group.

Two participants who withdrew on day 4 were not included in this analysis.

There were no early parasitological or clinical failures on days 1–3. Parasite clearance times (based on blood smears taken twice in a day) were shorter for participants assigned to artemether plus lumefantrine than for those assigned to AQ-13 (p=0·002; figure 2). By contrast, fever clearance times were shorter for those randomly assigned to AQ-13 than for those assigned to artemether plus lumefantrine (p=0·01; appendix). When the α used to identify significant differences was adjusted for multiple tests (table 2), parasite clearance times remained significantly shorter for those given artemether plus lumefantrine than for those given AQ-13 (figure 2), whereas the fever clearance times were significantly shorter for AQ-13 than for artemether plus lumefantrine (appendix). Although two participants in the AQ-13 group withdrew from the study on day 4, both had received five capsules of AQ-13 (1596 mg base) on days 1–3 and had cleared all asexual parasites on day 3. Likewise, the three participants lost to follow-up from the AQ-13 group on days 10, 28, and 35 had cleared all asexual parasites on day 3. By contrast, two participants in the artemether plus lumefantrine group had recurrences on days 17 and 21. Because the parasites causing these recurrences had the same molecular markers as the original infections in these participants, they were considered recrudescences (late treatment failures) rather than new infections: RO33 allotype parasites for participant 2010, and K1 and MAD20 allotype parasites for participant 2033.

Figure 2

Kaplan-Meier plot for parasite clearance times

Proportion of participants with positive blood smears after treatment with artemether and lumefantrine versus AQ-13.

Pharmacokinetic studies for the 17 participants randomly assigned to receive AQ-13 in 2015 identified separate peaks for each AQ-13 dose on days 1, 2, and 3 (figure 3). Mean blood concentration of AQ-13 8 days after the start of treatment was 800 nM, which is about 40–50 times higher than the 15–20 nM IC50 of AQ-13 for chloroquine-resistant parasites. Blood concentration data are provided only for AQ-13 because the metabolites of AQ-13 (AQ-72 and AQ-73) are not active against chloroquine-resistant P falciparum. Table 3 shows pharmacokinetic parameters for AQ-13 based on blood samples from the 17 participants randomised to receive AQ-13 in 2015. Neither the 1-week area under the curve, elimination half-life, time from beginning treatment to the maximal concentration, peak maximal concentration, nor other pharmacokinetic parameters correlated with physical parameters such as height, weight, or body-mass index (data not shown).

Figure 3

Mean blood concentrations of AQ-13 after oral treatment from days 1 to 8

Mean blood concentrations of AQ-13 after treatment with 1596·25 mg of AQ-13 base on days 1–3 are based on serial 5·0 mL venous blood samples from 17 participants randomly assigned to and given AQ-13 in 2015. Error bars are 95% CIs. The mean AQ-13 blood concentrations observed 7–8 days after the start of treatment (800–1000 nM) were 40–50-times greater than the IC50s for chloroquine-resistant Plasmodium falciparum in vitro (15–20 nM).7, 8 IC50s=50% inhibitory concentrations.

Table 3

Pharmacokinetic data for participants randomly assigned to and given AQ-13

	Peak Cmax (μM)	Time to peak tmax(h)	1-week AUC (μM/h)	MRT (days)	Cl/f (L/h)	Elimination t½(days)
Participant ID number 2034	3·637	30·0	273·2	4·29	4·52	0·91
Participant ID number 2036	1·399	30·0	162·3	14·47	4·95	13·35
Participant ID number 2038	3·231	25·0	197·2	3·10	7·48	1·49
Participant ID number 2040	2·865	24·5	213·0	7·30	4·49	4·54
Participant ID number 2042	2·585	28·0	175·4	5·17	6·15	3·01
Participant ID number 2043	2·042	28·0	208·4	14·04	3·57	8·23
Participant ID number 2046	3·457	30·0	274·2	6·17	3·89	3·92
Participant ID number 2047	3·992	28·0	353·3	5·88	3·23	3·67
Participant ID number 2050	2·313	28·0	193·3	4·26	6·64	1·67
Participant ID number 2051	2·591	28·0	210·1	7·85	4·35	1·55
Participant ID number 2052	2·280	28·0	252·7	6·71	3·98	4·30
Participant ID number 2055	1·722	30·0	158·6	4·61	7·95	3·68
Participant ID number 2058	2·793	28·0	183·8	5·25	6·13	3·16
Participant ID number 2061	2·152	30·0	157·4	5·44	7·35	4·12
Participant ID number 2062	1·572	28·0	128·9	4·95	9·21	2·16
Participant ID number 2064	1·407	26·0	110·4	4·61	11·23	2·53
Participant ID number 2066	2·900	28·0	239·7	5·29	4·57	3·11
Mean (95% CI)	2·526 (2·16–2·89)	28·1 (27·3–28·9)	205·4 (176·9–233·9)	7·19 (5·20–9·18)	5·86 (4·81–6·91)	3·85 (2·44–5·26)

Data are of 17 participants who were randomly assigned to receive AQ-13 in 2015 in the pharmacokinetic studies. Cmax=maximal concentration. tmax=time from beginning treatment to the maximal concentration. 1-week AUC=area under the curve for the first 7 days. MRT=mean residence time. Cl/f=clearance. t½=elimination half-life.

On the basis of the eye examinations, there were no differences in visual acuity between the groups (table 2). Only 31 (93·9%) of 33 participants given AQ-13 had the follow-up eye exam on day 7 because two participants withdrew from the study on day 4.

Holter recordings showed that all participants were in normal sinus rhythm at screening and enrolment, and none had atrial or ventricular arrhythmias during the inpatient stay (days 1–4) or the outpatient visits on days 7 and 14. Likewise, none had first, second, or third degree heart block during the inpatient stay or the outpatient follow-up visits (table 2). One participant was excluded from the study because of heart failure with ventricular bigeminy. This patient had uncomplicated P falciparum malaria and was referred to the senior cardiologist after receiving artemether with lumefantrine. No significant increases in the QTc interval were observed after treatment with either artemether plus lumefantrine or AQ-13 (data not shown). Additionally, comparison of QTc intervals at the time of peak AQ-13 blood concentrations 4 h after dosing for artemether plus lumefantrine and for AQ-13 showed no differences (table 2).

No serious, grade 2, grade 3, or grade 4 adverse events were identified in participants given either artemether plus lumefantrine or AQ-13. Because some adverse events were expected and had been reported with the same dose of AQ-13 in the phase 1 study, participants were asked about potential adverse events daily during the 1-week inpatient stay and at each outpatient follow-up visit. Several less serious adverse events (≤grade 1) were reported by participants (table 4). Of the 453 less severe adverse events (≤grade 1) reported, those associated with acute malaria (fever, weakness, myalgias and arthralgias, headache, anorexia, nausea, vomiting, and abdominal pain) were most common on days 1–3 (158 events per day), less common on days 4–6 (11 events per day), and least common on days 7–28 (three events per day). There were no significant differences in the frequencies of less serious adverse events for individuals between the treatment groups.

Table 4

Less serious adverse events (≤grade 1)

	Artemether plus lumefantrine (n=33)	AQ-13 (n=33)	p value
Fever	29 (88%)	32 (97%)	0·36
Weakness	28 (85%)	27 (82%)	1·00
Myalgias and arthralgias	25 (76%)	27 (82%)	0·76
Headache	31 (94%)	32 (97%)	1·00
Anorexia	20 (61%)	24 (73%)	0·43
Nausea	13 (39%)	17 (52%)	0·46
Vomiting	7 (21%)	12 (36%)	0·28
Abdominal pain	8 (24%)	9 (27%)	1·00
Diarrhoea	6 (18%)	1 (3%)	0·11
Cough	8 (24%)	7 (21%)	1·00
Pruritus	9 (27%)	16 (48%)	0·13
Tinnitus	2 (6%)	5 (15%)	0·43
Influenza-like syndrome	11 (33%)	9 (27%)	0·79
Temperature	13 (39%)	16 (48%)	0·62
Pallor	2 (6%)	3 (9%)	1·00
Jaundice	2 (6%)	2 (6%)	1·00

Data are the numbers of participants (%) reporting each adverse event (different from number of reports for each adverse event because the same adverse event was often reported more than once for an individual person).

Pruritus had a different temporal pattern to the other adverse events. Reports of pruritus were most frequent on days 4–6 (nine events per day), less frequent on days 1–3 (six events per day), and least frequent on days 7–28 (0·2 events per day). However, there were no differences in the frequency of pruritus between the groups (p=0·13; table 4). The 1·5 g/dL decrease in haemoglobin concentration between days 1 and 4 in participants receiving artemether plus lumefantrine had not been expected (table 2). The decrease in haemoglobin concentration was greater in those given artemether plus lumefantrine than in those given AQ-13 (1·5 g d/L [95% CI 0·7–2·3] vs 0·4 g/dL [0·3–0·5]; p=0·02), although both were less than the grade 1 decrease on the AIDS grading scale (2·5–3·4 g/dL). When the α used to identify significant differences was adjusted for multiple tests, the decrease in haemoglobin concentration after treatment was no longer greater for artemether plus lumefantrine than for AQ-13 (data not shown).

Discussion

Our per-protocol analysis showed that AQ-13 was non-inferior to artemether plus lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria. However, in the intention-to-treat analysis, AQ-13 was inferior to artemether with lumefantrine, because of dropouts from the AQ-13 group.

The five AQ-13 failures in the intention-to-treat analysis were in participants lost to follow-up rather than true treatment failures, and we identified no cases in which AQ-13 treatment did not clear the parasite. Because the differences between per-protocol and intention-to-treat analyses were caused by more participants being lost to follow-up in the AQ-13 group, enrolling more participants might have eliminated the discrepancies. However, we followed the recommendations of the US Food and Drug Administration to enrol the fewest participants necessary (to minimise the number exposed to candidate compounds that might not be efficacious).

The efficacy of artemether plus lumefantrine for the treatment of uncomplicated P falciparum malaria (≥95%)11, 21 and the absence of efficacy data for AQ-13 favoured a non-inferiority study design. However, because AQ-13 is a single agent and artemether plus lumefantrine is a combination therapy, the clinical outcomes might have been better for artemether plus lumefantrine than for AQ-13, even if AQ-13 was also efficacious because of the potential for complementary or synergistic interactions with drug combinations.

The similar ages, parasite densities, haemoglobin concentrations, and frequencies of the T76 and K76 aminoacid point mutations (CVIET and CVMNK haplotypes) in both groups indicate there were no detectable differences in outcome related to pfcrt and suggest that randomisation distributed potential confounding factors successfully. The recrudescences (late treatment failures) reported with artemether plus lumefantrine were consistent with the 5% or less failure rate reported for this treatment.13, 21 By contrast, no recrudescences were reported with AQ-13.

When the α used to identify significant differences was adjusted for multiple tests, the rate of parasite clearance remained significantly quicker for artemether plus lumefantrine than for AQ-13. However, neither the faster fever clearance with AQ-13 nor the greater fall in haemoglobin concentration after treatment with artemether plus lumefantrine remained significant. As a result, these data do not resolve those questions (ie, what is the frequency of haemolysis after artemisinin vs AQ-13 treatment of uncomplicated malaria? And what is the relative parasite and fever clearance rates for artemisinins vs AQ-13?) because this study was not powered to address them. However, previous studies have found faster parasite clearance with artemether plus lumefantrine than with other antimalarial drugs, faster fever clearance with treatment regimens containing 4-AQ, and haemolysis after the treatment of severe malaria with artemisinins.

The absence of serious, grade 2, grade 3, and grade 4 adverse events in either group is consistent with data indicating serious adverse events are uncommon with artemether plus lumefantrine13, 21, 24, 25 and with AQ-13, although experience with AQ-13 is limited. As in the phase 1 study, several participants reported less serious (≤grade 1) adverse events with AQ-13, in particular for pruritus. Although pruritus has been associated with the use of 4-AQs, it has also been reported in people treated with artemether plus lumefantrine, artesunate plus sulfadoxine and pyrimethamine, and other antimalarial drugs.21, 25 For this reason, our observations are consistent with reports by other investigators on pruritus after antimalarial treatment. Therefore, generalised pruritus might be caused by antimalarial drugs that are different structurally from 4-AQs or else pruritus might occur in patients with malaria whether they are or are not treated with antimalarial drugs.

The decrease in haemoglobin concentration after treatment with artemether plus lumefantrine is consistent with reports of haemolysis after artesunate treatment for severe malaria, and suggests post-artemisinin haemolysis might also occur in patients with less severe (uncomplicated) malaria, although this finding was not significant after adjusting for multiple tests. On the basis of these observations, we suggest adding post-treatment haemolysis to the list of potential adverse events associated with artemisinins in uncomplicated malaria. By contrast, the decrease in haemoglobin concentration with AQ-13 was consistent with the haemolysis typically reported in uncomplicated P falciparum malaria.

Changes in visual acuity have been reported with chloroquine and hydroxychloroquine, but usually only in patients treated for a decade or more. Likewise, arrhythmias and heart block have been reported with chloroquine and hydroxychloroquine, but typically only after massive intravenous overdoses administered rapidly rather than the low oral doses administered in our study. No ocular or cardiac adverse events were reported in either treatment group.

The more rapid parasite clearance time with artemether plus lumefantrine than with AQ-13 was expected because many studies have reported shorter clearance times with artemisinins. However, the shorter fever clearance times with AQ-13 than with artemether plus lumefantrine were not expected. These results suggest that fever clearance is not simply a secondary effect of parasite clearance. Additionally, the shorter fever clearance times with AQ-13 were consistent with reports that 4-AQs have antipyretic activity. Thus, the antipyretic activity of the 4-AQs might be responsible for the shorter fever clearance times reported with AQ-13 despite the shorter parasite clearance times reported with artemether plus lumefantrine.

AQ-13 concentrations were 40–50 times greater than the IC50 for drug-resistant parasites, and were maintained in the blood for 8 days or more as asexual parasites were cleared without serious adverse events. Of note, the pharmacokinetic parameters and blood concentrations of AQ-13 in this phase 2 study are similar to those observed in the phase 1 study of AQ-13 in New Orleans, LA, USA, which enrolled healthy men and women who did not have malaria or other acute illnesses. We did not detect a measurable prolongation of the QTc interval with AQ-13, despite the findings of the phase 1 study. Because the AQ-13 blood concentrations were similar in our study and the phase 1 study, we postulate that AQ-13 sequestered within infected red blood cells might not be free to affect the QT interval.

The data reported in our study indicate that the safety and efficacy of AQ-13 for uncomplicated malaria are similar to those of artemether plus lumefantrine in Malian men aged 18 years or older. However, because semi-immune participants, such as those included in our study, might clear parasites resistant to the drugs being tested, additional studies in non-immune individuals, such as children, are needed before deciding whether to recommend the widespread use of aminoquinolines such as AQ-13. Other limitations of this study include the exclusion of women and the exclusion of individuals with severe or complicated malaria.

Because of increasing resistance to artemisinin combination therapies in southeast Asia, the efficacy of AQ-13 for uncomplicated malaria caused by chloroquine-resistant P falciparum and the activity of other aminoquinolines against chloroquine-resistant parasites7, 8 suggest that 4-AQs with modified side chains could improve malaria control in sub-Saharan Africa, Latin America, and Oceania, and possibly in southeast Asia.